Objective
To investigate if a synthetic, biodegradable scaffold with either autologous in vitro cultured muscle-derived cells or autologous fresh muscle fiber fragments could be used for tissue repair.
Study Design
Twenty scaffolds with muscle-derived cells and 20 scaffolds with muscle fiber fragments were implanted subcutaneously on the abdomen of rats, 2 in each rat, and examined after 3 weeks (10 of each preparation) and 8 weeks (10 of each preparation). Immonohistochemistry and histopathology was undertaken for assessment of growth pattern and biocompatibility, respectively.
Results
At 3 weeks, both muscle-derived cells and muscle fiber fragments could be identified. At 8 weeks, the muscle fiber fragments generated fragmented, striated muscle tissue in 6 of 10 explants, whereas the muscle-derived cells and all scaffolds had vanished.
Conclusion
Autologous fresh muscle fiber fragments on a biodegradable scaffold seem useful for tissue repair. This study introduces a promising new concept with possible implications for the surgical reconstruction of pelvic organ prolapse.
The use of synthetic, permanent implants (meshes) for reinforcement in the surgical reconstruction of pelvic organ prolapse (POP) has become popular in order to improve outcome. However, documentation regarding efficacy is tenuous, whereas complications, such as erosions, shrinkage, and dyspareunia, have emerged. In 2008, the US Food and Drug Administration (FDA) issued a Public Health Notification regarding the serious complications associated with transvaginal placement of implants in the treatment of POP and stress urinary incontinence ( www.fda.gov ).
The discipline of regenerative medicine includes cell-based tissue repair, and muscle-derived cells seeded on biodegradable scaffolds have been proposed recently in the treatment of POP to avoid the complications associated with permanent, synthetic implants. Ho et al succeeded in stimulating vaginal repair in rats using murine muscle-derived stem cells seeded on scaffolds made from porcine small intestine submucosa (SIS). Animal-derived scaffolds, however, have drawbacks, such as limited availability, high cost, variable host tissue response, and concerns for disease transmission. This makes a synthetic, biodegradable scaffold like methoxypolyethyleneglycol-poly(lactic-co-glycolic acid) (MPEG-PLGA) more appealing.
Increasing regulatory demands ( www.ema.europa.eu ; www.fda.gov ) practically impede cost-effectiveness and, consequently, the clinical relevance of using in vitro cultured stem cells in the treatment of benign disorders. Fresh muscle fiber fragments collected at the time of surgery may offer a more effective approach. This technique will not be restricted by these regulations, and have been used experimentally in other areas of regenerative medicine.
In this study, we investigated in a rat model if MPEG-PLGA with either autologous cultured muscle-derived cells or autologous muscle fiber fragments could be used for tissue repair.
Materials and Methods
The animal experiments were conducted at the Animal Facility at the Panum Institute, Copenhagen. Approval was obtained from the institutional review board (the Danish Animal Experiments Inspectorate) with permission no. 2009/561-1585, and their guidelines for the care and use of the animals were followed.
The experimental animals were Sprague Dawley retired female breeder rats weighing 300-435 g (Taconic, Ejby, Denmark). The Panum Institute provided animal housing and caretaking.
Scaffolds were made of MPEG-PLGA (Coloplast, Humlebaek, Denmark), a degradable and more hydrophilic material than the PLGA known as Vicryl (Ethicon Inc., Somerville, NJ), and with a high degree of biocompatibility and cellular in-growth. MPEG-PLGA is also structurally different from Vicryl and other knitted implants through its spongy structure with interconnected pores ( Figure 1 ).
Two different preparations of the scaffold were used: (1) scaffold with autologous muscle-derived cells, primarily myoblasts, which are 1 of several stages of muscle progenitor cells, and (2) scaffold with autologous fresh muscle fiber fragments.
Implantation
Each type of scaffold was tested in 10 rats for 3 weeks and in another 10 rats for 8 weeks. The rat abdominal subcutaneous model allowed for the testing of 2 pieces of scaffold per rat.
Rats were anesthetized with Hypnorm/Midazolam 0.3 mL/100 g (Hypnorm; Veta Pharma Ltd., Leeds, UK, and Midazolam; Hameln Pharmaceuticals GmbH, Hameln, Germany). A 4-cm midline incision was made on the abdomen. After subcutaneous blunt dissection, the scaffolds measuring 10 × 20 × 1 mm were placed superficially to the abdominal muscle fascia and tacked in position with one stitch of Vicryl 4-0. Scaffolds were placed longitudinally to the midline. The skin was closed with a continuous suture of Vicryl 4-0, and the knots were hidden subcutaneously in both ends to avoid self-mutilation from the rats. Antibiotic prophylaxis and pain medications were administered according to veterinarian recommendations. Rats were euthanized at 3 and 8 weeks after implantation.
Muscle biopsies
A 2-cm incision was made on the hind leg of the rat, and 2 muscle biopsies were obtained using a biopsy punch of 4 mm diameter. The skin was closed as described previously.
Scaffolds with muscle-derived cells
Muscle biopsies were obtained 2 weeks before abdominal implantation. Biopsies were immediately transferred to containers with transport medium and left overnight at 4°C for further processing (see below).
Scaffolds with muscle fiber fragments
Muscle biopsies were obtained immediately before the abdominal implantation. The muscle fiber fragments were prepared in a sterile Petri dish with 2 scalpels by cutting the biopsies into a fine mash in physiologic saline (1 drop). The scaffold was added, and the muscle fiber fragments instantly attached to it. At implantation, the muscle fiber fragment-covered side of the scaffold faced the fascia.
Isolation and culture of muscle-derived cells
Muscle-derived cells were grown at Interface Biotech A/S, Hoersholm, Denmark. Isolation of muscle-derived cells was done according to a modification of “Gene Delivery to Muscle”-protocol, which primarily results in myoblasts as verified by desmin stain. In brief, the biopsies were minced thoroughly; 0,5 mL collagenase/dispase/CaCl 2 was added and mincing continued; the mixture was incubated at 37°C for 1 hour; centrifuged for 5 minutes at 350×g at room temperature; the supernatant was removed; cells were resuspended in 10 mL F-10 based culture medium and plated in collagen-coated flasks. Cells were seeded in 25 cm 2 flasks.
After 7 days of culture, the cells were trypsinized and transferred to collagen-coated flasks. After another 7 days of culture, cells were trypsinized, counted, and seeded in a concentration of 2 × 10 6 cells per scaffold. Cells were seeded on the scaffolds 24 hours implantation, and the scaffolds with cells were incubated overnight and shipped to the animal facility.
Explantation
Scaffolds with surrounding full-thickness host tissue were harvested, fixed in 10% buffered formalin, and routinely processed for immunohistochemistry and histopathology. The thickness of the sections was 5 μm.
Immunohistochemistry
The growth pattern of muscle-derived cells and muscle fiber fragments was assessed by immunohistochemical staining. To identify skeletal muscle cells as opposed to smooth muscle cells, 2 different primary antibodies were used: monoclonal mouse antihuman desmin (1:100, Clone D33; DAKO, Glostrup, Denmark) and monoclonal mouse antihuman α-smooth muscle actin (SMA) (1:100, Clone 1A4; DAKO). The known cross-reactive specificity of the antibodies with equivalent proteins in rats was confirmed by positive and negative controls. Desmin stains the cytoplasm of both skeletal and smooth muscle cells, whereas SMA stains the cytoplasm of only smooth muscle cells. It was necessary to pretreat the paraffin embedded sections to unmask the antigens. For desmin, we used heat-induced epitope retrieval in target retrieval solution, High pH (S2367, diluted 1:10; DAKO), whereas for SMA, we used proteolytic treatment with pepsin solution (Digest-All 3 [Pepsin]; Invitrogen, Taastrup, Denmark). The incubation time for both of the primary antibodies was overnight at 4°C. The sections were incubated 10 minutes at room temperature with the secondary antibody (Histostain-Plus Kit; Invitrogen). 3-amino-9-ethylcarbazole (AEC) or DAB (3,3′-diaminobenzidine) was used as chromogen for visualization. Cell nuclei were counterstained with hematoxylin.
One section of each 3-week specimen was stained for each primary antibody, and the correct location was ensured microscopically by the scaffold being present. If an 8-week-section was negative for desmin with regard to muscle-derived cells or muscle fiber fragments, additional 6 sections, interspaced 30 μm, were stained to ensure the absence of muscle-derived cells or muscle fiber fragments in nearby foci.
Histopathology
All explants were stained with hematoxylin and eosin (H&E), Giemsa (Sigma-Aldrich, St. Louis, MO), and Masson’s Trichrome (Sigma-Aldrich).
Biocompatibility assessed from the degree of inflammation and vascularity was scored in a semi-quantitative manner on a scale of 0–4, as described previously. At 3 weeks, the assessment was done within the scaffold. At 8 weeks, where the scaffold had disappeared, assessment was done within the remaining granulation tissue at the site. Host foreign body response due to the suture material was noted but not evaluated.
All histologic assessments were done in cooperation with a senior pathologist (L.C.) blinded to the type of implant.
Data regarding immunohistochemistry are presented as absolute numbers and frequencies by percent. Data from histopathologic assessment are presented as medians and ranges, and analyzed using the nonparametric Mann–Whitney U test (SAS version 9.1; SAS Institute, Cary, NC). P values < .05 were considered statistically significant.
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